Identification and functional characterization of a pyrokinin neuropeptide receptor in the Lyme disease vector, Ixodes scapularis

Gondalia, Kinsi; Qudrat, Anam; Bruno, Brigida A.; Medina, Janet Fleites; Paluzzi, Jean‐Paul

doi:10.1016/j.peptides.2016.09.011

Cited by 21 publications

(23 citation statements)

References 68 publications

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“…Unlike previous reports showing GPA2/GPB5-induced LGR1 activation elevates intracellular cAMP by coupling a Gs pathway, the current findings provide novel information supporting that A. aegypti LGR1 couples to a Gi protein to inhibit cAMP levels following application of heterodimeric GPA2/GPB5. Further, our results revealed that mosquito LGR1 is constitutively active when overexpressed in the absence of its ligand, GPA2/GPB5, inducing a Gs signalling pathway that raises levels of cAMP levels, which is consistent with previous observations with overexpression of fruit fly LGR1 29, 51 as well as mammals including dog and human TSH receptor 52, 53 .…”

Section: Discussionsupporting

confidence: 92%

Expression Profiling, Downstream Signaling and Subunit Interactions of GPA2/GPB5 in the Adult MosquitoAedes aegypti

Rocco

Paluzzi

2019

Preprint

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GPA2/GPB5 and its receptor constitute a glycoprotein hormone-signalling system native to the genomes of most vertebrate and invertebrate organisms, including humans and mosquitoes. Unlike the well-studied gonadotropins and thyrotropin, the exact function of GPA2/GPB5 is unclear, and whether it elicits its functions as heterodimers, homodimers or as independent monomers remains unknown. Here, the glycoprotein hormone signalling system was investigated in adult mosquitoes, where GPA2 and GPB5 subunit transcripts co-localized to bilateral pairs of neuroendocrine cells within the first five abdominal ganglia of the central nervous system. Unlike human GPA2/GPB5 that demonstrated strong heterodimerization between subunits, the GPA2/GPB5 subunits in A. aegypti lacked evidence of heterodimerization when heterologously expressed. Interestingly, cross-linking analysis to determine subunit interactions revealed A. aegypti and H. sapiens GPA2 and GPB5 subunits form homodimers, and treatments with independent subunits did not activate A. aegypti LGR1 or H. sapiens TSH receptor, respectively. Since mosquito GPA2/GPB5 heterodimers were not evident by heterologous expression of independent subunits, a tethered construct was generated for expression of the subunits as a single polypeptide chain to improve heterodimer formation. Our findings revealed A. aegypti LGR1 elicited constitutive activity that elevated levels of cAMP as determined by increased cAMP-dependent luminescence. However, upon treatment with recombinant tethered GPA2/GPB5 heterodimers, an inhibitory G protein (Gi) signalling cascade is initiated and forskolin-induced cAMP production is inhibited. These results provide evidence towards the functional deorphanization of LGR1 and, moreover, further support the notion that GPA2/GPB5 heterodimerization is a requirement for glycoprotein hormone receptor activation.

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Section: Discussionsupporting

confidence: 92%

Expression Profiling, Downstream Signaling and Subunit Interactions of GPA2/GPB5 in the Adult MosquitoAedes aegypti

Rocco

Paluzzi

2019

Preprint

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“…Most information on tick GPCRs was obtained from recombinant receptor systems (Yang et al, 2013, 2015; Gross et al, 2015; Gondalia et al, 2016; Kim et al, 2018). Transcripts for tick GPCRs are often expressed in vivo at very low levels, which complicates their physiological characterization.…”

Section: Discussionmentioning

confidence: 99%

The Cattle Fever Tick, Rhipicephalus microplus, as a Model for Forward Pharmacology to Elucidate Kinin GPCR Function in the Acari

2019

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The success of the acaricide amitraz, a ligand of the tick tyramine/octopamine receptor (a G protein-coupled receptor; GPCR), stimulated interest on arthropod-specific GPCRs as targets to control tick populations. This search advances tick physiology because little is known about the pharmacology of tick GPCRs, their endogenous ligands or their physiological functions. Here we explored the tick kinin receptor, a neuropeptide GPCR, and its ligands. Kinins are pleiotropic insect neuropeptides but their function in ticks is unknown. The endogenous tick kinins are unknown and their cDNAs have not been cloned in any species. In contrast, more than 271 insect kinin sequences are available in the DINeR database. To fill this gap, we cloned the kinin cDNA from the cattle fever tick, Rhipicephalus microplus , which encodes 17 predicted kinins, and verified the kinin gene structure. We predicted the kinin precursor sequences from additional seven tick species, including Ixodes scapularis . All species showed an expansion of kinin paracopies. The “kinin core” (minimal active sequence) of tick kinins FX 1 X 2 WGamide is similar to those in insects. Pro was predominant at the X 2 position in tick kinins. Toward accelerating the discovery of kinin function in ticks we searched for novel synthetic receptor ligands. We developed a dual-addition assay for functional screens of small molecules and/or peptidomimetics that uses a fluorescent calcium reporter. A commercial library of fourteen small molecules antagonists of mammalian neurokinin (NK) receptors was screened using this endpoint assay. One acted as full antagonist (TKSM02) with inhibitory concentration fifty (IC 50 ) of ∼45 μM, and three were partial antagonists. A subsequent calcium bioluminescence assay tested these four antagonists through kinetic curves and confirmed TKSM02 as full antagonist and one as partial antagonist (TKSM14). Antagonists of NK receptors displayed selectivity (>10,000-fold) on the tick kinin receptor. Three peptidomimetic ligands of the mammalian NK receptors (hemokinin 1, antagonist G, and spantide I) were tested in the bioluminescence assay but none were active. Forward approaches may accelerate discovery of kinin ligands, either as reagents for tick physiological research or as lead molecules for acaricide development, and they demonstrate that selectivity is achievable between mammalian and tick neuropeptide systems.

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“…Total RNA (50 ng) was then used as template for cDNA synthesis using the iScript TM Reverse Transcription Supermix (Bio-Rad, Mississauga, ON, Canada) following recommended guidelines and diluted ten-fold prior to quantitative RT-PCR. LGR1 transcript levels were then quantified using PowerUP TM SYBR ® Green Master Mix (Applied Biosystems, Carlsbad, CA, United States) and measured on a StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, United States) following conditions described previously (Gondalia et al, 2016). Primers used to quantify LGR1 transcript abundance overlap exon-exon boundaries (Supplementary Table S1), and data was normalized to the stably expressed reference gene ribosomal protein 49 (GenBank accession: AY539746) according to the ΔΔCt method (Paluzzi et al, 2014).…”

Section: Methodsmentioning

confidence: 99%

Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti

et al. 2019

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Glycoprotein hormone receptors mediate a diverse range of physiological functions in vertebrate and invertebrate organisms. The heterodimeric glycoprotein hormone GPA2/GPB5 and its receptor LGR1, constitute a recently discovered invertebrate neuroendocrine signaling system that remains to be functionally characterized. We previously reported that LGR1 is expressed in the testes of adult Aedes aegypti mosquitoes, where its immunoreactivity is particularly regionalized. Here, we show that LGR1 immunoreactivity is associated with the centriole adjunct of spermatids and is observed transiently during spermatogenesis in mosquitoes, where it may act to mediate the regulation of flagellar development. RNA interference to downregulate LGR1 expression was accomplished by feeding mosquito larvae with bacteria that produced LGR1-specific dsRNA, which led to defects in spermatozoa, characterized with shortened flagella. LGR1 knockdown mosquitoes also retained ∼60% less spermatozoa in reproductive organs and demonstrated reduced fertility compared to controls. To date, the endocrine regulation of spermatogenesis in mosquitoes remains an understudied research area. The distribution of LGR1 and detrimental effects of its knockdown on spermatogenesis in A. aegypti indicates that this heterodimeric glycoprotein hormone signaling system contributes significantly to the regulation of male reproductive biology in this important disease-vector.

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Identification and functional characterization of a pyrokinin neuropeptide receptor in the Lyme disease vector, Ixodes scapularis

Cited by 21 publications

References 68 publications

Expression Profiling, Downstream Signaling and Subunit Interactions of GPA2/GPB5 in the Adult MosquitoAedes aegypti

Expression Profiling, Downstream Signaling and Subunit Interactions of GPA2/GPB5 in the Adult MosquitoAedes aegypti

The Cattle Fever Tick, Rhipicephalus microplus, as a Model for Forward Pharmacology to Elucidate Kinin GPCR Function in the Acari

Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti

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